Sequential requirements of the N-terminal palmitoylation site and SH2 domain of Src family kinases in the initiation and progression of FcεRI signaling
Sequential requirements of the N-terminal palmitoylation site and SH2 domain of Src family kinases in the initiation and progression of FcεRI signaling
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DOI：
10.1128/mcb.20.5.1759-1771.2000
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发表时间：
2000-03-01
影响因子：
5.3
通讯作者：
Yamamoto, K
中科院分区：
文献类型：
--
作者：
Honda, ZI;Suzuki, T;Yamamoto, K
源自高亲和力免疫球蛋白 E 受体 (Fc epsilon RI) 的最初生化信号传导被归因于 Src 家族激酶。为了了解单个激酶驱动信号传导的机制，我们进行了重建实验：RBL2H3细胞中的Fc epsilon RI信号传导首先被膜锚定的、功能获得性C端Src激酶抑制，然后用Src家族激酶重建，其C端负调节序列被c-myc表位取代。这些源自 Lyn 和 Fyn 的构建体与耐去污剂膜 (DRM) 相关，与静息的 Fc epsilon RI 发生物理相互作用，并重建簇诱导的信号传导，从而导致钙动员和 ERK1 和 -2 激活。 c-Src衍生的构建体被排除在DRM之外，无法与Fc epsilon RI相互作用并恢复信号传导，而棕榈酰化Cys3的产生使其能够与DRM和Fc epsilon RI相互作用并恢复信号传导。从 Lyn 衍生的构建体中删除 Src 同源 3 (SH3) 结构域并不会改变其转导一系列信号传导的能力。 SH2结构域的删除不影响其与DRM和Fc epsilon RI的关联，也不影响Fc epsilon RI beta和gamma亚基的聚类诱导的酪氨酸磷酸化，但它几乎取消了Syk的下一步酪氨酸磷酸化及其向Fc epsilon RI的募集。这些发现表明，Lyn 和 Fyn 可以驱动 Fc epsilon RI 信号传导，但 c-Src 不能，并且 N 末端棕榈酰化和 SH2 结构域在与 Fc epsilon RI 的初始相互作用以及信号进展至分子组装的序列中是必需的。
Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (Fc epsilon RI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: Fc epsilon RI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting Fc epsilon RI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with Fc epsilon RI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with Fc epsilon RI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with Fc epsilon RI nor clustering-induced tyrosine phosphorylation of Fc epsilon RI beta and gamma subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to Fc epsilon RI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive Fc epsilon RI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with Fc epsilon RI and for the signal progression to the molecular assembly.